Epidermal growth factor (EOF)receptor (EGFR) is important for the development and progression of many human epithelial cancers, associated with poor prognosis and has become the major prognostic marker and therapeutic target in a variety of these cancers. Elucidation of the physiological regulation of EGFR is, therefore, a key to understanding of the mechanisms causing its oncogenic activation and impairing the potential of EGFR therapeutics. Growth factor binding to the EGFR triggers a complicated array of signal transduction processes. Receptor activation also causes rapid endocytosis via clathrin-coated pits and degradation of the ligand-receptor complexes in lysosomes. Thus, endocytic trafficking determines the number of active EGFRs in the cell and,therefore, the intensity and duration of signaling processes. Endocytosis also plays role in spatial and temporal regulation of EGFR signaling, which may control the dynamics and the specific pattern of signaling processes. However, the mechanisms of EGFR endocytosis and its cross-talks with signaling processes in normal and cancer cells remain poorly understood. The main goal of this proposal is to delineate the mechanisms of EGFR endocytosis, and to define the role of endocytosis in the development and progression of cancer. During the previous funding cycle of this project we have identified key proteins that control the physiological pathway of clathrin-mediated internalization of EGFR. Our studies also demonstrated localization of EGFR signaling complexes in endosomes and the importance of endocytosis for signaling. We have developed novel quantitative methods of analysis of EGFR complexes in living cells and a novel methodology of the functional analysis of EGFR endocytosis using highly efficient RNA interference. These important advances make it now feasible to develop a comprehensive model of how EGFR endocytosis is regulated in cancer cells and how endocytic processes regulate cancerigenesis. In this project we focus on the analysis of head-and-neck cancer (HNC) where EGFR is frequently overexpressed and considered to be an important therapeutic target. The specific aims are: 1) Define the molecular mechanisms of EGFR endocytosis through clathrin coated pits. This aim will, in particular, define the role of ubiquitylation in EGFR endocytosis using novel methods of mass-spectrometry; 2) Test whether new EGFR mutants with impaired endocytic trafficking produce altered patterns of signaling in HNC cells in vitro and affect tumorigenic properties of HNC xenografts in vivo; 3) Test whether altered expression of proteins regulating endocytosis of EGFR and possibly other oncogenic receptor tyrosine kinases affect EGFR signaling in vitro and tumorigenic properties of HNC xenografts in vivo. The successful completion of the aims of the proposal will open new avenues to design of therapeutic and preventive strategies in treatment of EGFR expressing cancers.